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Mutation types

Alterao na sequncia nucleotdica

Vrias classsificaes
Tipo de clula: somtica ou linha germinal
Tipo de alterao molecular
Efeito fenotpico (na funo)
Agentes qumicos (mutagneos)
Agentes fsicos

Sistemas de reparao
Mutation might also occur during DNA replication


Two basic classes of mutations: somatic and
germ-line mutation

Nonreproductive cells Mosaics

Reproductive cells
Three basic molecular types of gene mutations are
base substitutions, insertions and deletions
Base substitution leads to two types of molecular change:
transition and tranversion

Base substitution during replication leads to two types of molecular change:

- transition
- tranversion
Codons that can result from a single base change in
tyrosine codon UAU
Different types of mutations caused by
base substitutions in coding regions

Normal Unpredictble Incomplete Normal

protein protein function protein protein

Ponctual mutations
Molecular basis of sickle-cell anemia. Consequences of
base substitution example- missense mutation

The resulting hemoglobin is defective and tends to polymerize at low oxygen concentration
Insertion/deletion of a nucleotide
Frameshift mutation
Other classifications for phenotypic effects of

Loss-of-function (null or knockout) (eliminates normal


Gain-of-function (ectopic expression) (expressed at

incorrect time, or in appropriate cell types)

Hypomorphic (leaky) (reduces normal function, usually

due to low level gene expression)

Hypermorphic (increases normal function, usually due

to high level gene expression)
Terminologia de mutao
(frequentemente aplicada a microorganismos)

Auxotrofos- no cresce em meio mnimo porque a

mutao afecta um gene que codifica uma molcula
biolgica essencial

Constituitivos- expresso (transcrio) permanente de

um gene. Ex. regio do operador do opero lac

Condicionais- ex. mutaes termosensveis (Ts)- s se

manifestam sob determinadas condies (no

Letais condicionais- mutaes que s se manifestam

sob determinadas condies, e quando tal so letais

Incondicionais- manifestam-se sob condies

permissivas e no permissivas
Relation of foward, reverse and supressor
An INTRAGENIC supressor mutation occurs in the
same gene that contains the mutation being
Model for the effect of mutation
and intragenic supression on
the folding and activity
An INTERGENIC supressor mutation occurs in a different gene
from the one bearing the original mutation

Mutation in a different gene

Leu tRNA gene

Mutant Leu tRNA gene


Mutant Leu tRNA

Efeitos das mutaes
Mutaes silenciosas
No DNA intergnico
Em regies no codificantes
Numa base do tripleto, sem alterar o aa

Mutaes em regies codificantes


Mutaes em regies no codificantes, mas no silenciosas

Regio do promotor
Sequncias reguladoras
Origem replicao
Limiar exo/intro ou mesmo no intro
Novos locis de splicing alternativo
Spontaneous mutation
(in absence of known mutagen)
Induced mutation
(in presence of known mutagen)
Spontaneous chemical changes



Deamination (may also be induced by mutagenic

(tautomeric forms)

keto enol

keto enol

amino imino
-NH2 =NH

amino imino
Pairing relationships of DNA bases in the normal and tautomeric

Tautomeric shifts results in transition mutations. The tautomerization can occur in the:
- template base, ie, tautomerization of the base in the template
- substrate base, ie, tautomerization of incoming base.

Perda do grupo amina NH2

Espontnea ou induzida
Deamination: spontaneous loss of
amino group

Methylated cytosine
Methylation of cytosine at the number-5 position in the
base. The methyl donor is S-adenosylmethyonine
Mechanism by which uracil-containing nucleotides
are formed in DNA and removed (E. coli)

Uracil is cleaved from the deoxyribose sugar by DNA uracil glycosylase

The deoxyribose with the uracil detached is then excised from the DNA backbone
by another enzyme (AP endonuclease) and the gap is repaired

Deamination of 5-methylcytosine leads to a mutation

5MeC G TG Replication T A (mutant)

G C (wt)
Chemical induced mutations

Chemical environmental agents that significantly

increase the rate of mutation above the spontaneous

Base analogs (ex. 5-Bu, 2-AP)
Chemicals that alter bases
Nitrous acid- deamination
Alkylating agents (EMS, NTG, nitrogen mustards, mitomycin C)
Intercalating agents (EtBr, proflavin )
Reactive forms of oxygen (ex superoxide radicals)- oxidative
Base analogues

Principal mechanism of mutagenesis of base analogs: increased rate on base mispairing

Mispairing mutagenesis by 5-bromouracil

Normal pairing Mispairing

Nucleotide analogue

AZT is used in the clinical treatment of AIDS

Chemicals may alter DNA bases
Highly mutagenic alkylating agents

The effect of alkylation depends on the

position at which the nucleotide is modified and the type
of of alkyl group that is added.
Alkylation may alter
base-pairing properties
and so lead to point mutations,
or cause structure distortion forming crosslinks between the two strands, blocking replication.
Principal mechanism of mutagenesis: bulky attachments made to side groups on bases
Adenine deamination due to nitrous acid

Altered A pairs with C



Pu-Py Pu-Py

Insert between adjacent bases in DNA, distorting the three-dimensional

structure of the helix and causing single-nucleotide insertions and
deletions in replication
Physical agents

Ionizing radiation
In the electromagnetic spectrum, as wavelenght decreases, energy increases

Ionizing radiation


Pyrimidime dimers result from ultraviolet light

Different types of bonds between

the thymine rings are also possible

Distortion of the DNA helix

DNA replication and transcription are blocked
Ionizing radiation

Source: x-rays, radon gas, radioactive


Mechanism of mutagenesis:
single and double-stranded breaks in DNA
damage to nucleotides
Tcnicas de Mutagnese

Aleatria (random)

Chemical mutagenesis
using sodium bisulfite

Transio: C-G T-A

Oligonucleotide-directed mutagenesis by
enzymatic primer extension

Plasmid DNA is isolated from the resulting colonies

and is screened to identify mutants
Enrichment for oligonucleotide-directed
mutants by using a uracil-containing template

Single-stranded DNA is prepared in a ung- dut-

E. coli strain
ung - DNA uracil glycosylase deficient
dut - dUTPAse deficient (high levels of dUTP)

Following ligation, the heteroduplex DNA molecules

are introduced in a ung+ E. coli strain
Quick-Change site directed mutagenesis

DNA isolated from most E. coli strains

is dam methylated

DpnI- is specific for methylated and

hemimethylated DNA
Mutation Repair
Sistemas de reparao
Directos (no substituem o nt alterado, mas repem a sua estrutura original)

Fotoreparao enzimtica. Ex. fotoliase de E. coli

Remoo enzimtica de grupos qumicos que se ligam s bases

dos nts e os alteram. Ex enzima ADA de E. coli que remove os grupos alquilo na posio 6 da guanina

DNA ligase que actua sobre cortes em cadeia simples (nicks)

DNA polimerase I e DNA ligase (E. coli) que actuam em lacunas


Recombino homloga em gaps ou cortes em cadeia dupla

Sistemas de reparao (cont.)
Exciso de bases e nts

Glicosilases (enzimas especficas de re+arao do DNA). Ex. uracil

glicosilase (ung). Geram locais apurnicos (Depurinao)

Endonucleases AP- removem o acar-fosfato nos locais apurnicos (AP)

Exciso de nucletiodos pelo sistema MutHLS (geralmente associado a um

incorrecto emparelhamento de bases- mismatch)

Exciso de nucletidos devido a bases modificadas que distorcem a

configurao normal do DNA. Ex. dmeros de timina, bases alteradas do cido
a ligao de grupos qumicos)- Sistema UvrABC

Sistema SOS (E. coli)

DNA clivado em ambas as cadeias (protenas

Ku70 e Ku80 + cinase de DNA + )
Direct repair: enzymatic removal
changes nucleotides back into their original stuctures

- ADA in E.coli
- MGMT (O6-methylguanine-DNA methyltransferase)
in humans
Base and nucleotide excision repair
Excises modified bases and then
replaces the entire nucleotide

Each DNA glycosylase enzyme

recognizes and removes a specific
type of damaged base, producing
an apurinic or an apyrimidinic site
(AP site)

The endonuclease AP cleaves

the phosphodiester bond on
the 5 side of the AP site and
removes the deoxyribose sugar

Just after DNA replication Many incorrectly inserted nucleotides that
escape proofreading are corrected by
MutHLS - mismatch repair

The mismatch is brought close to a

methylated GATC sequence, and
the new strand is identified

Helicase and single-stranded

exonuclease remove nucleotides
on yhe new strand between the
GATC sequence and the mismatch

DNA polymerase I, DNA ligase

Dam methylase
Excision repair of DNA by
E. coli UvrABC mechanism

UvrA/UvrB complex detect

conformational changes in DNA
Helix to become locally denatured
and kinked by 130

UvrC endonuclease binds and

cuts the damaged strand at two
sites separated by 12 or 13 bases

Helicase II unwinds the damaged region,

releasing the single-stranded fragment
with the lesion, which is degraded to

The gap is filled by DNA polymerase I, and

the remaining nick is sealed by DNA ligase